Great demand for novel pain therapies exists for chronic pain states. The arachidonic acid (ARA) cascade is a pivotal inflammatory pain pathway, and many studies addressed the cyclooxygenase (cox) and lipoxygenase (lox) branches of this cascade. However, the emerging importance of a third branch, the cytochrome P450 pathway, offers novel alternatives for pain modulation. In contrast to the inflammatory cox and lox metabolites, the cytochrome P450-generated ARA metabolites, epoxyeicosatrienoic acids (EETs), are potent physiological anti-inflammatory/antihyperalgesic molecules. These bioactive lipids have very short half lives and inhibiting their degradation mediated by soluble epoxide hydrolase (sEH, EC 3.3.2.3) leads to antinociception. Inhibitors of sEH are effective in models of inflammatory and neuropathic pain, but it is unclear how sEH inhibition attenuates pain, warranting further investigation of this novel pathway. The sEH is well expressed in the peripheral and central nervous system, in the neurons and the glial cells, with spatial selectivity of expression among brain regions. Inhibitors of sEH (sEHIs) block nocifensive behavior, while leaving intact normal nociceptive and motor function. Thus in this study we will test the hypothesis that epoxygenated fatty acids and sEHI have a modulatory role in pain pathophysiology. To address this hypothesis, the levels of the EpFAs and their degradation products in the skin and the lumbar spinal cord in two distinct models of pain will be monitored. Levels of EpFAs will then be modulated in two distinct models of pain by different approaches; a) by exogenous EpFAs, b) by inhibiting the sEH, c)by inhibiting phosphodiesterases and d) by novel synthetic analogues of EpFAs that are resistant to sEH mediated degradation. We will utilize the carrageenan induced inflammatory pain model to identify anti- hyperalgesic EpFAs. The bioactivity of anti-nociceptive EpFAs will be validated in a clinically relevant model, the incisional pain model. We will use a combination of behavioral assays, biochemical and metabolomic analysis to understand the interactions of EpFAs with nociceptive pathways. We will produce a characteristic/diagnostic LC/MS/MS generated metabolomic profile of the intraplantar carrageenan and the plantar incisional pain models in rats. The incisional pain in particular is less well studied in regard to alterations in bioactive lipid metabolites. The target metabolites (80+ bioactive lipids) are from all three branches of the AA cascade including major cox, lox, cytochrome P450 and the sEH products including lipid metabolites of EPA and DHA, allowing us to investigate metabolite flux and crosstalk between biological cascades. The proposed studies will result in an advanced understanding of the antinociceptive mechanism of action of EpFAs and sEH inhibitors, and provide novel insights to the underlying mechanisms of lipidomic and biochemical alterations in response to pain. The knowledge generated by these experiments is likely to lead to the development of novel classes of agents that target pathways that have not been recognized previously.